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Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions ("quellung" effects). A problem in studying Ab-capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab-capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system.

Allergic bronchopulmonary mycosis (ABPM) is a hypersensitivity lung disease in which fungal colonization is accompanied by an allergic response to the fungus. Using a mouse model of ABPM caused by Cryptococcus neoformans infection of C57BL/6 mice, the goal of the present studies was to determine the effect of the CD4-depleting monoclonal antibody GK1.5 on the development of the allergic responses seen during active fungal infection. These results would provide insight into the role of CD4(+) T cells in this disease. Our results show that GK1.5 treatment resulted in attenuation of pulmonary inflammation and eosinophilia in these animals. These mice also had reduced T2 cytokine production and no serum immunoglobulin E production. Absence of CD4(+) T cells did not affect recruitment of CD8(+) T cells to the site of infection; however, the numbers of CD19(+) B cells were severely reduced in the lungs of CD4(+) T-cell-depleted animals. We also examined changes in the pulmonary architecture and found that depletion of CD4(+) T cells was associated with a significant reduction in mucus production and goblet cell metaplasia in these mice. Interestingly, attenuation of Th2 responses in CD4(+) T-cell-depleted animals did not increase the fungal load in their lungs. We also compared development of ABPM in young and mature mice and did not find any differences at any of the time points. Overall, our results show that depletion of CD4(+) T cells prevents the development of Th2 responses seen during ABPM.

Killer toxin-neutralizing monoclonal antibody (nmAb-KT) against HM-1 killer toxin (HM-1) produced by yeast Williopsis saturnus var. mrakii IFO 0895 reduces both the killing and glucan synthase inhibitory activity of HM-1. nmAb-KT is classified as IgG1kappa and has been shown to be ineffective against HYI killer toxin produced by the related yeast W. saturnus var. saturnus IFO 0117. To determine the epitope for nmAb-KT, overlapping peptides were synthesized from the primary structure of HM-1. nmAb-KT reacted with peptides P5 (33NVHWMVTGGST43), P6 (39TGGSTDGKQG48) and P7 (44DGKQGCATIWEGS56), which represent the middle region of the HM-1 sequence. P6 reacted most strongly with nmAb-KT. Combined analysis by immunoblotting, surface plasmon resonance (SPR) analysis and yeast growth inhibition assay showed that nmAb-KT recognizes a specific epitope within peptide P6. The K(d) value of nmAb-KT against HM-1 and P6 were determined to be 5.48 x 10(-9) M and 1.47 x 10(-6) M by SPR analysis, respectively. These results strongly indicate that nmAb-KT binds to HM-1 at the sequence 41GSTDGK46, and not to HYI at the same position. The potential active site of HM-1 involved in the killing activity against sensitive yeast is discussed.

The protective efficacy of mAbs to Cryptococcus neoformans glucuronoxylomannan depends on Ab isotype. Previous studies in A/JCr and C57BL/6J mice showed relative protective efficacy of IgG1, IgG2a >> IgG3. However, we now report that in C57BL/6J x 129/Sv mice, IgG3 is protective while IgG1 is not protective, with neither isotype being protective in 129/Sv mice. IgG1, IgG2a, and IgG3 had different effects on IFN-gamma expression in infected C57BL/6J x 129/Sv mice. IgG1-treated C57BL/6J x 129/Sv mice had significantly more pulmonary eosinophilia than IgG2a- and IgG3-treated C57BL/6J x 129/Sv mice. C. neoformans infection and Ab administration had different effects on FcgammaRI, FcgammaRII, and FcgammaRIII expression in C57BL/6J, 129/Sv, and C57BL/6J x 129/Sv mice. Our results indicate that the relative efficacy of Ab isotype function against C. neoformans is a function of the genetic background of the host and that IgG3-mediated protection in C57BL/6J x 129/Sv mice was associated with lower levels of IFN-gamma and fewer pulmonary eosinophils. The dependence of isotype efficacy on host genetics underscores a previously unsuspected complex relationship between the cellular and humoral arms of the adaptive immune response.

Both CD4(+) T cells and B cells are critical for defense against Pneumocystis carinii infection; however, the mechanism by which B cells mediate protection is unknown. We show that P. carinii-specific IgM is not sufficient to mediate clearance of P. carinii from the lungs since CD40-deficient mice produced normal levels of specific IgM, but were unable to clear the organisms. Using chimeric mice in which the B cells were deficient in CD40 (CD40KO chimeras) we found that clearance of P. carinii infection is delayed compared with wild-type controls. These CD40KO chimeric mice produced normal levels of P. carinii-specific IgM, but did not produce class-switched IgG or IgA. Similarly, clearance of P. carinii was delayed in mice deficient in FcgammaRI and III (FcgammaRKO), indicating that P. carinii-specific IgG partially mediates opsonization and clearance of P. carinii. Opsonization of organisms by complement did not compensate for the lack of specific IgG or FcgammaR, since C3-deficient and C3-depleted FcgammaRKO mice were still able to clear P. carinii. Finally, micro MT and CD40KO chimeric mice had reduced numbers of activated CD4(+) T cells in the lungs and lymph nodes compared with wild-type mice, suggesting that B cells are important for activation of T cells in response to P. carinii. Together these data indicate that P. carinii-specific IgG plays an important, but not critical, role in defense against P. carinii. Moreover, these data suggest that B cells also mediate host defense against P. carinii by facilitating CD4(+) T cell activation or expansion.

Protective immunity to Candida albicans and Aspergillus fumigatus is mediated by antigen-specific Th1 cells. To define the role of B cells and antibodies in the generation of antifungal immune resistance, B cell-deficient (mu MT) mice were assessed for immune resistance to primary and secondary infections with both fungi. The results showed that, although passive administration of antibodies increased the fungal clearance, the innate and Th1-mediated resistance to the primary and secondary infections were both heightened in mu MT mice with candidiasis and aspergillosis. However, although capable of efficiently restricting the fungal growth, mu MT mice did not survive the re-infection with C. albicans, and this was concurrent with the failure to generate IL-10-producing dendritic cells and regulatory CD4(+)CD25(+) T cells. Antifungal opsonizing antibodies restored IL-10 production by dendritic cells from mu MT mice, a finding suggesting that the availability of opsonizing antibodies may condition the nature of the dendritic cell interaction with fungi, possibly impacting on the development of long-lasting antifungal immunity.

We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2(-/-)) and the parental strain. Parental mice lived significantly longer than NOS2(-/-) mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2(-/-) and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2(-/-) mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2(-/-) mice resulted in significantly higher levels of IFN-gamma, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha than parental mice. Ab administration had different effects on infected NOS2(-/-) and parental mice with respect to IFN-gamma, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha levels. Ab administration increased lung levels of IFN-gamma in parental mice and reduced levels in NOS2(-/-) mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.

A murine mAb (mAbKT4, IgG1) that neutralized in vitro the anti-Candida activity of a killer toxic (KT) from the yeast Pichia anomala acted as an idiotypic (Id) vaccine in eliciting anti-Id Abs with toxin-like activity (KT-IdAb) in a rat vaginitis model. In this study, we demonstrate that intravaginal or intragastric inoculations of Candida albicans bearing a receptor for the toxin was able to recall KT-IdAb production in the vagina of the animals primarily immunized with mAbKT4 and also to elicit by themselves an Ab that functionally mimicked the KT (KTAb). Anti-Id-like, KT-like Abs were also consistently found in the vaginal fluid of human vaginitis patients who were infected by Candida but who had never been exposed to the Id vaccine. These Abs were as candidacidal in vitro as those raised in rat vagina by the Id vaccination, and, likewise, their cytocidal effect was totally neutralized by previous reaction with mAbKT4. Importantly, they were also able to confer a significant anticandidal protection in the rat vaginitis model, comparable to that achievable by KT-IdAb passively transferred to naive rats from Id-vaccinated animals. Thus, candidacidal Abs representing the internal image of a yeast KT are part of the Ab repertoire that follows infection or immunization with Candida. It is speculated that the host's immune system response may exploit the KT receptor of microbial pathogens to produce microbicidal Abs, possibly mirroring competition events among microorganisms in natural habitats.

In this paper we relate that sera from paracoccidioidomycosis patients inhibited the mitogen-induced proliferative responses of normal mononuclear cells. Treatment of these sera with 2.5% polyethyleneglycol (PEG), a method classically used to precipitate immune complexes, significantly reduced their inhibitory activity. Immunoblot analysis of the PEG precipitates identified a 34-kDa polypeptide, recognized by rabbit anti-P. brasiliensis IgG. Patient mononuclear cells showed partial restoration of their proliferative capacity after 24 h culture in medium alone, which suggests release of membrane-bound molecules in the culture medium. These findings indicate that circulating P. brasiliensis antigens, complexed or not with antibodies, may play a negative immunoregulatory effect in the mitogen-induced proliferative responses of paracoccidioidomycosis patients.

B-cell-deficient mice were prepared by administration of rabbit anti-mouse-mu antiserum to newborn animals within 12 h of birth onwards. Such immunodeficient animals, along with the normal controls, were infected intravenously with Cryptococcus neoformans. There was no difference in the mortality pattern, viable count of cryptococci in different organs, delayed-type hypersensitivity reaction, and antigen level in the sera of control and B-cell-deficient animals. Antibodies were absent in B-cell-deficient animals but were present in low titers in control animals. It is concluded that antibodies are not involved in protection of mice infected with C. neoformans.

Following intranasal inoculation of mice, Rhizomucor pusillus spores are gradually cleared from the lung, with the clearance complete at 30 days. Incubation of R. pusillus spores in vivo for up to 14 days after intranasal inoculation resulted in pulmonary mucormycosis when the mice were then treated with cortisone. Spore-agglutinating IgM antibodies were detected in normal adult mice and R. pusillus-inoculated but not cortisone-treated mice. There was no correlation between antispore antibody titers and spore germination in vitro. The absence of germinated R. pusillus in inoculated but non-cortisone-treated mice appears to be due to a reversible inhibition of spore germination rather than destruction of spores by the host. The factor(s) that restrict spore germination are reversible and do not appear to be spore agglutinating antibodies.

The influence of preformed antibody on the induction of experimental Candida albicans endocarditis was investigated by both in vitro and in vivo techniques. Preincubation of C. albicans with immune serum (raised in rabbits by intravenous injection of Formalin-killed yeast cells) decreased adhesion to the constituents of nonbacterial thrombotic endocarditis, e.g., fibrin plus platelets, in vitro. Two different methods, with radiolabeled or viable yeast cells, were confirmatory and demonstrated decreased adhesion of immune serum-treated C. albicans cells to 0 to 7.8% of control values (P less than 0.001). These results correlated with protection from the development of C. albicans endocarditis in the immunized rabbits. The mean (+/- standard deviation) infectious dose for 50% of the animals was 10(5.29) +/- 10(0.07) in 48 control animals versus 10(7.11) +/- 10(0.22) in 37 immunized rabbits (P less than 0.001). These studies suggest that humoral antibody may protect against C. albicans endocarditis, perhaps through inhibition of adhesion, a crucial early step in the pathogenesis of endocarditis.